The study of Chlamydia is complicated by the lack of a genetic transformation system. The development of such a system will likely revolutionize research in this area. This proposal describes an approach to solving this problem using Chlamydia suis as a target for gene transfer. The experiments are based on the recent discovery of a stable tetracycline resistance island within the genome of recent isolates of C. suis from pigs in production facilities. This island is structurally related to plasmids of Gram-negative bacteria, and was likely introduced to this species via horizontal transmission from an unknown bacterial species. Genes within the island encode plasmid mobilization and replication functions, tetracycline efflux pump, and candidate transposases. Experimental data in a surrogate system suggests that one of these transposases was responsible for the integration of the genomic island into C. suis following its introduction as a plasmid. It is hypothesized that the encoded transposase and the tet(C) gene can be used as tools to introduce genes into Chlamydia suis and for screening possible positives, respectively. This proposal describes different approaches that will be explored to test this hypothesis, with the ultimate goal of developing a workable genetic system for the chlamydiae. First, a novel screening technique under development in our laboratory will be optimized for isolating candidate tetracycline-resistant transfromants from a population of cells infected with tetracycline-sensitive C. suis. Next, recombinant plasmids will be constructed that represent the likely donor plasmid responsible for the natural introduction of DNA into C. suis. These plasmids will be used in several different techniques designed to introduce the DNA into the bacterium. Several variations of possible transformation and conjugation-mediated approaches will be used in these attempts, all based on the premise that introduced DNA will serve to deliver the tet(C) island to the chlamydial chromosome. If a technique for chlamydial transformation is developed, we will expand the work into a five-year proposal that will address the application of the technology and the transfer of the protocol to investigators in the field. [unreadable] Success in the proposed studies will lead to opportunities for rapid developments in our understanding of chlamydial biology. This will assist in the identification and analysis of novel vaccine candidates and to a clearer understanding of the mechanism of virulence in these important pathogens. [unreadable] [unreadable] [unreadable] [unreadable]